生物学杂志 ›› 2021, Vol. 38 ›› Issue (5): 59-.doi: 10.3969/j.issn.2095-1736.2021.05.059

• 研究报告 • 上一篇    下一篇

不同碳源细胞自噬对巴斯德毕赤酵母生长代谢的调控

  

  1. 1.江南大学 粮食发酵工艺与技术国家工程实验室, 无锡 214122,
    2. 江南大学 生物工程学院, 无锡 214122
  • 出版日期:2021-10-18 发布日期:2021-10-21
  • 通讯作者: 杨艳坤,副教授,硕士生导师,主要从事微生物学、分子生物学、发酵工程研究,E-mail:yangyankun@jiangnan.edu.cn;白仲虎,教授,博士硕士导师,主要从事发酵工程、分子生物学研究,E-mail:baizhonghu@jiangnan.edu.cn
  • 作者简介:陆文怡,硕士,主要从事发酵工程、分子生物学研究,E-mail:lillianlwy@126.com
  • 基金资助:
    国家自然科学基金项目(21908077,31570034);江苏省第十五批六大人才高峰项目(SWYY-180)

Regulation of autophagy on the growth and metabolism of Pichia pastoris using different carbon sources

  1. 1. National Engineering Laboratory of Cereal Fermentation Technology, Jiangnan University, Wuxi 214112, China;
    2. School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2021-10-18 Published:2021-10-21

摘要: 为挖掘不同碳源影响毕赤酵母(Pichia pastoris)生长代谢的机制,对其进行了比较研究。结果发现,相较于甘油,当碳源转变为甲醇时,细胞内宏自噬相关基因(Autophagy associated gene 8, Atg8)的表达量显著上升,而过氧化物酶自噬相关基因(Autophagy associated gene 30, Atg30)的表达量下降。甲醇培养时,细胞内宏自噬水平提高,菌体生长缓慢,而过氧化物酶体自噬被抑制,AOX1酶的活性提高。同时,对比甘油转运体(Glycerol transporter 1, GT1)敲除的菌株(P. pastoris ΔGT1)与野生型菌株(P. pastoris Wild Type, WT)的细胞自噬受碳源调控的规律,发现甲醇和甘油引起的P. pastoris ΔGT1细胞内Atg8和Atg30基因表达变化的差值低于野生型菌株。说明GT1基因的敲除缓解了甘油和甲醇对细胞自噬调控的差异,这也符合P. pastoris ΔGT1菌株缓解了甘油抑制甲醇代谢的特性,进一步证实自噬水平的调控是不同碳源引起细胞生长代谢改变的主要原因之一。从细胞自噬角度解析了巴斯德毕赤酵母碳源代谢调控的机制,同时为进一步提高甲醇利用效率和外源蛋白表达效率提供了新的方向和理论基础。

关键词: 毕赤酵母, 生长代谢, 过氧化物酶体自噬, 宏自噬, 甘油转运体

Abstract: Pichia pastoris has become one of the most popular eukaryotic expression systems for its strong promoter of alcohol oxidase Ⅰ (AOX1), PAOX1. However, the growth of the cell in methanol (short for MeOH) is obviously slower compared to glycerol. And glycerol can inhibit methanol metabolism. In order to clarify the mechanism how carbon sources affect yeast growth and metabolism, the effects of different carbon sources on Pichia pastoris were compared. All experimental results showed that the change of metabolic substrate and cell growth were involved in autophagy. When the carbon source was changed to methanol, the expression of Atg8 was up-regulated, while Atg30 was down-regulated. In methanol, higher level of macroautophagy resulted in slower cells growth, inhibited pexophagy and increased activity of AOX1. The change of autophagy in Glycerol transporter 1 knockout Pichia pastoris (short for P. pastoris ΔGT1) was compared with that in wild-type and found that the change of the expression level of Atg8 and Atg30 was smaller in P. pastoris ΔGT1, which conformed to the property that carbon source suppression was released in P. pastoris ΔGT1. It was further demonstrated that the autophagy was one of the main ways that carbon sources changed the cell growth and metabolism. This paper further analyzed the mechanism of methanol metabolism regulation of Pichia pastoris from the perspective of cell autophagy, and provided a new direction and theory to improve utilization of methanol and expression of heterogeneous proteins.

Key words: Pichia pastoris, growth and metabolism, pexophagy, macroautophagy, glycerol transporter

中图分类号: